The activity of BK (slol) channels in brain artery myocytes and neurons is modified by acute exposure to ethanol (EtOH) levels reached in blood during alcohol intoxication, such modification contributing to EtOH perturbation of physiology. EtOH actions in the body may require drug-mediated potentiation or inhibition of BK channels. Our long-term goal is to identify molecular mechanisms and protein sites that lead to BK channel differential responses to EtOH and their contribution to drug actions on the brain. This knowledge is essential towards identification of brain vulnerability and design of therapeutic interventions in alcohol-driven pathology via BK channel-targeting. In particular, EtOH inhibition of cerebral artery myocyte BK current is the key event in alcohol-mediated cerebral artery constriction, an alcohol action that is independent of circulating and endothelial factors, and likely contributes to alcohol-induced cerebrovascular disease and accidents. The myocyte-abundant BK pi subunit (coded by KNCMB1) facilitates slol (coded by KCNMA1) channel inhibition by EtOH and coupling to type2 ryanodine receptors (RyR2), these proteins constituting a triad that controls cerebral artery tone and is directly targeted by EtOH. Aim1 addresses the role of BK p i membrane levels in EtOH vulnerability of brain arteries that irrigate distinct CNS regions and are differentially subject to cerebrovascular events. The aim is tested in rodent models (rat, KCNMB1 K/0 mouse) using both in vitro (Western-blotting, protein biotinylation, ICC/IF microscopy, patch-clamp, cDNA reverse-permeabilization, and isolated myocytes and arteries) and in vivo methods (closed cranial window, intravital microscopy). Defined by similar models and methods, and supplemented by computational modeling and medicinal chemistry, Aim2 determines whether BK pi-targeting by novel agents antagonizes cerebral artery constriction caused by EtOH. Putative EtOH-sensing sites and regions in the three proteins that constitute the EtOH target: BK pi (SubAim 3.1), slol (SubAim 3.2) and RyR2 (Aim 4) are identified via computational modeling, mutagenesis and in vitro electrophysiology, the impact of these sites on physiology being established by reverse-permeabilization of relevant rodent artery (KCNMB1 K/O, KCNMA1 K/O mice). RELEVANCE (See instructions): Cerebral artery constriction is often a main component of brain conditions and accidents associated with ethanol intoxication. We will identify the ethanol-recognition sites in the ion channels that mediate ethanol-induced cerebral artery constriction and use this information to point at vulnerability and therapeutics to counteract cerebrovascular pathophysiology caused by ethanol intake.